We report that a subset of avian genes is characterized by very high GC content and long G/C stretches. These sequence characteristics correlate with the frequent absence of these genes from genomic databases. We provide several examples where genes in this subset are mistakenly reported as missing in birds.www.dx.doi.org/10.1186/s13059-015-0725-y

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine playing critical roles in host defense and acute and chronic inflammation. It has been described in fish, amphibians, and mammals but was considered to be absent in the avian genomes. Here, we report on the identification and functional characterization of the avian ortholog. The chicken TNF-α (chTNF-α) is encoded by a highly GC-rich gene, whose product shares with its mammalian counterpart 45% homology in the extracellular part displaying the characteristic TNF homology domain. Orthologs of chTNF-α were identified in the genomes of 12 additional avian species including Palaeognathae and Neognathae, and the synteny of the closely adjacent loci with mammalian TNF-α orthologs was demonstrated in the crow (Corvus cornix) genome. In addition to chTNF-α, we obtained full sequences for homologs of TNF-α receptors 1 and 2 (TNFR1, TNFR2). chTNF-α mRNA is strongly induced by lipopolysaccharide (LPS) stimulation of monocyte derived, splenic and bone marrow macrophages, and significantly upregulated in splenic tissue in response to i.v. LPS treatment. Activation of T-lymphocytes by TCR crosslinking induces chTNF-α expression in CD4⁺ but not in CD8⁺ cells. To gain insights into its biological activity, we generated recombinant chTNF-α in eukaryotic and prokaryotic expression systems. Both, the full-length cytokine and the extracellular domain rapidly induced an NFκB-luciferase reporter in stably transfected CEC-32 reporter cells. Collectively, these data provide strong evidence for the existence of a fully functional TNF-α/TNF-α receptor system in birds thus filling a gap in our understanding of the evolution of cytokine systems.

Since the publication of the first chicken genome sequence, we have encountered genes playing key roles in mammalian immunology, but being seemingly absent in birds. One of those was, until recently, Foxp3, the master transcription factor of regulatory T cells in mammals. Therefore, avian regulatory T cell research is still poorly standardized. In this study we identify a chicken ortholog of Foxp3. We prove sequence homology with known mammalian and sauropsid sequences, but also reveal differences in major domains. Expression profiling shows an association of Foxp3 and CD25 expression levels in CD4⁺CD25⁺ peripheral T cells and identifies a CD4⁻CD25⁺Foxp3^high subset of thymic lymphocytes that likely represents yet undescribed avian regulatory T precursor cells. We conclude that Foxp3 is existent in chickens and that it shares certain functional characteristics with its mammalian ortholog. Nevertheless, pathways for regulatory T cell development and Foxp3 function are likely to differ between mammals and birds. The identification and characterization of chicken Foxp3 will help to define avian regulatory T cells and to analyze their functional properties and thereby advance the field of avian immunology. Key Points Foxp3 is present in the chicken genome. Domain organization and synteny are largely conserved. Phenotypically, chicken regulatory T cells resemble their mammalian counterparts.

The ongoing progress in primordial germ cell derivation and cultivation is opening new ways in reproductive biotechnology. This study tested whether functional sperm cells can be matured from genetically manipulated primordial germ cells after transplantation in adult testes and used to restore fertility. We show that spermatogenesis can be restored after mCherry-expressing or GFP-expressing primordial germ cells are transplantated into the testes of sterilized G₀ roosters and that mCherry-positive or GFP-positive non-chimeric transgenic G₁ offspring can be efficiently produced. Compared with the existing approaches to primordial germ cell replacement, this new technique eliminates the germ line chimerism of G₀ roosters and is, therefore, faster, more efficient and requires fewer animals. Furthermore, this is the only animal model, where the fate of primordial germ cells in infertile recipients can be studied.

The expression level of acute phase proteins (APPs) mirrors the health status of an individual. In human medicine, C-reactive protein (CRP), and other members of the pentraxin family are of significant relevance for assessing disease severity and prognosis. In chickens, however, which represent the most common livestock species around the world, no such marker has yet gained general acceptance. The aim of this study was therefore, to characterize chicken pentraxin 3 (chPTX3) and to evaluate its applicability as a general marker for inflammatory conditions. The mammalian and chicken PTX3 proteins were predicted to be similar in sequence, domain organization and polymeric structure. Nevertheless, some characteristics like certain sequence sections, which have varied during the evolution of mammals, and species-specific glycosylation patterns, suggest distinct biological functions. ChPTX3 is constitutively expressed in various tissues but, interestingly, could not be found in splenic tissue samples without stimulation. However, upon treatment with lipopolysaccharide (LPS), PTX3 expression in chicken spleens increased to 95-fold within hours. A search for PTX3 reads in various publicly available RNA-seq data sets of chicken spleen and bursa of Fabricius also showed that PTX3 expression increases within days after experimental infection with viral and bacterial pathogens. An experimental infection with avian pathogenic E.coli and qPCR analysis of spleen samples further established a challenge dose-dependent significant up-regulation of chPTX3 in subclinically infected birds of up to over 150-fold as compared to untreated controls. Our results indicate the potential of chPTX3 as an APP marker to monitor inflammatory conditions in poultry flocks.

Significance: Retroviruses copy their RNA genome into complementary DNA, which is then inserted into the host chromosomal DNA as an obligatory part of their life cycle. Such integrated viral sequences, called proviruses, are passed to the infected cell progeny on cellular division. If germline cells are targeted, the proviruses become vertically inherited as other host genes and are called endogenous retroviruses. Deltaretroviruses, which include important human and veterinary pathogens (HTLV-1 and BLV), are the last retroviral genus for which endogenous forms were not known. We have identified a case of endogenous Deltaretrovirus, which entered the genome of long-fingered bat ancestors more than 20 million years ago. This finding opens the way for elucidating the deep evolutionary history of deltaretroviruses.